Annex 3 Guidelines to assure the quality, safety and efficacy of live attenuated rotavirus vaccines (oral)

Transcription

1 World Health Organization WHO Technical Report Series No 941, 2007 Annex 3 Guidelines to assure the quality, safety and efficacy of live attenuated rotavirus vaccines (oral) Introduction General considerations Special considerations Use of primary cells for the production of rotavirus vaccine Part A. Guidelines on manufacturing A.1 Defi nitions A.2 General manufacturing recommendations A.3 Control of source materials A.4 Control of vaccine production A.5 Filling and containers A.6 Control tests on fi nal product A.7 Records A.8 Samples A.9 Labelling A.10 Distribution and shipping A.11 Storage and expiry date Part B. Nonclinical evaluation of live attenuated rotavirus vaccines Part C. Clinical evaluation of live attenuated rotavirus vaccines Part D. Guidelines for national regulatory authorities D.1 General D.2 Release and certifi cation Authors Acknowledgements References Appendix 1 Summary protocol of manufacturing and control of live attenuated rotavirus vaccines (oral) Appendix 2 Genealogy of vaccine production process Appendix 3 Model certifi cate for the release of live attenuated rotavirus vaccines 133

2 This document provides guidance to national regulatory authorities and vaccine manufacturers on the production, quality control and evaluation of safety and efficacy of live attenuated rotavirus vaccines (oral) by outlining, in detail, international regulatory expectations for product characterization. It should be read in conjunction with the WHO guidelines on nonclinical evaluation of vaccines (1), and the WHO guidelines on clinical evaluation of vaccines: regulatory expectations (2), in order to understand the whole process of vaccine evaluation. Advice that is specific to the nonclinical or clinical evaluation of live attenuated rotavirus vaccines is provided to supplement these two generic documents. The following text is written in the form of guidelines instead of recommendations. Guidelines allow greater flexibility than recommendations with respect to expected future developments in the field. Introduction The high incidence of rotavirus disease around the world has prompted international agencies including the World Health Organization (WHO), the Global Alliance for Vaccines and Immunization (GAVI), Children s Vaccine Program and the Rotavirus Vaccine Programme at the Program for Appropriate Technology in Health (PATH) to identify rotavirus vaccine development as one of their highest priorities. In response to this interest in the development of candidate rotavirus vaccines, WHO held an informal consultation on the Quality, Safety and Efficacy Specifications for Live Attenuated Rotavirus Vaccines in Mexico City, from 8 9 February 2005 (3). The experts present considered it timely for WHO to develop production and quality control guidelines and technical specifications for these vaccines. Draft guidelines on production and quality control specifications for live attenuated rotavirus vaccine (oral) were developed by a small drafting group established by WHO and were comprehensively reviewed by a WHO Informal Consultation on the proposed WHO Guidelines to Assure the Quality, Safety and Efficacy of Live Attenuated Rotavirus Vaccines held in Geneva, from August After taking into account the comments made during the consultation, the revised document was submitted to and approved by the WHO Expert Committee on Biological Standardization at its 56th meeting. The scope of this document covers live attenuated rotavirus vaccines (oral), and vaccine candidates, that are intended for international use. The aim of vaccination against rotavirus infection is to induce immunity against relevant rotavirus serotypes in one series of inoculations for the prevention of rotavirus gastroenteritis. The prevalence of multiple and different rotavirus serotypes throughout the world complicates vaccine development strategies. According to the data available in 2005, the majority of rotavirus disease 134

3 in humans is caused by five distinct serotypes of rotavirus, designated G1, G2, G3, G4 and G9 on the basis of the molecular composition of one outer coat viral protein (VP7). In the last decade, intense surveillance of rotavirus and improved laboratory assay methods have resulted in the detection of additional G serotypes that play a role in human disease. Although serotypes G1 G4 still predominate worldwide, serotypes G5, G8 and G9 have been isolated in Latin America, Africa and India respectively, and are beginning to cause a higher percentage of cases of the disease. The relative benefits of monovalent, multivalent or regional specific vaccines will remain unclear until efficacy data demonstrating heterotypic protection against relevant rotavirus serotypes become available. The scope of this document is restricted to live vaccines, but the properties of the possible candidates are varied, particularly with respect to their degree of attenuation. This affects the extent to which they grow in culture and in the human gut and applies to strains currently being investigated as well as those studied previously. There will therefore be major quality issues specific to a particular vaccine, such as the assay of vaccine potency, the stability of the virus in production, the yield of virus and the extent of contamination by cell-derived materials. Clinical issues which will vary between candidates include the dose required to obtain immunity in recipients, the possibility of transmission to contacts of vaccinees and the genetic stability of the virus on replication in the gut of recipients. Although many of the points of possible concern considered in this document are generally applicable to rotavirus vaccines, it must be remembered that each candidate must be examined individually and that this may raise significant product-specific issues. Several oral rotavirus vaccines are currently approved or in various stages of vaccine development. Each vaccine is the result of a unique approach in developing an attenuated phenotype and to the prevention of rotavirus disease. As more vaccines become readily available around the world, data will be provided to help in making decisions on the compositions of future vaccines. Examples of vaccines approved or in development include multivalent human bovine and human rhesus reassortant vaccines and monovalent vaccines containing a single attenuated human rotavirus strain; natural human bovine reassortants or animal rotavirus strains. Vaccines for which controlled efficacy studies have been completed have demonstrated high levels of efficacy (> 80%) against severe rotavirus gastroenteritis and have also shown a range of heterotypic protection against multiple serotypes. The impact of introduction of the vaccine on the epidemiology of natural rotavirus infection is as yet unknown. All of the vaccine candidates are claimed to be attenuated; however, the basis for the attenuation phenotype of the current vaccine candidates is 135

4 unknown. Typically the method of attenuation is to exploit the host range restriction properties of animal rotaviruses (Jennerian) by laboratory or natural (neonatal rotavirus strains) genetic reassortment with human rotavirus with the desired serotypes or insertion of multigenic mutations by serial passage in cell culture. Because laboratory markers of attenuation are not well defined and animal models demonstrating attenuation are not readily available, the claim of attenuation is based on clinical experience in human subjects. How the product will actually behave in clinical use is therefore based on clinical trials and consistency of production rather than on any specific laboratory tests. Potential laboratory markers of product consistency include information on the genetic sequence of the virus seed to show that a new seed material is similar to the previous seed and that each can be distinguished from the parent virus. Multiple passages of virus seed materials under defined cell culture conditions as well as examination of vaccine virus isolated from stool samples of vaccinees may be helpful to generate data on the conditions favouring genetic stability of the virus. Studies on consistency of production would need to take into account the variability inherent in replication of RNA virus and assess the presence of minority populations, as revealed for example by the occurrence of mixed base signals in sequencing studies. If minority populations are detected, it will be necessary to assess their biological importance, for example, by careful comparison of the level of heterogeneity between the master or working seed and higher passage levels e.g. clinical trial material. The development of any new rotavirus vaccines must take into account the events which led to the withdrawal of one vaccine (RotaShield) from the marketplace. RotaShield was introduced in the United States in August 1998 and was withdrawn less than 1 year later when an epidemiological relationship was established between RotaShield vaccination and intussusception (IS). Early estimates suggested a risk of one case per 2500 children immunized. Re-analysis of the case control study that examined intussusception and RotaShield revealed that the majority of the cases of IS associated with the first dose occurred in children 4 months of age or older. This did not comply with the manufacturer s recommendation that the first dose should be given at 2 months of age and thus changed the early estimates of attributable risk of IS in the target population (4). The detailed pathogenic mechanisms for IS are unclear, but are very likely to be complex. General considerations Considerable experience in the manufacturing, testing and clinical evaluation of rotavirus vaccines has been gained from over 20 years of their development. Candidate vaccines have been and are being studied in a number of countries with diverse economic conditions and geographical 136

5 boundaries. The regulatory approvals of two rotavirus vaccines and the submission for approval of another provide a solid foundation for the preparation of these guidelines and for the continuing development of new rotavirus vaccines; they also support the global introduction of rotavirus vaccines. These experiences have enabled standards to be set for vaccine efficacy, identified safety concerns, highlighted areas of scientific weakness, and led to the introduction of new manufacturing technologies and testing strategies. Issues relevant to the development of rotavirus vaccines are discussed below. Rotavirus is an acid-labile virus which is rapidly inactivated at an acid ph and has a half-life of less than 12 minutes at ph 2.0. Because rotavirus vaccines are intended to be administered to infants by the oral route, the virus would be inactivated by stomach gastric acid prior to reaching the site of infection in the upper gastrointestinal tract. To prevent inactivation of the virus by gastric acid, antacids or buffers are usually administered before or in combination with the oral rotavirus vaccination. The composition of the antacid and the mode of administration (in combination with vaccine or administered separately) will depend upon the biological characteristics of the vaccine virus. The processing of live rotavirus vaccines, like that of many other live viral vaccines, involves cell disruption and, if any, incomplete purification of the virus. In-process steps for the inactivation of adventitious agents are not included for live viral vaccines, as these steps may compromise the live nature of the vaccine itself. As a result, validation of clearance of any adventitious agents may not be possible. For these reasons a comprehensive set of tests for adventitious agents and qualification of the vaccine source materials is essential as part of the vaccine safety control. As with any viral vaccines, production of rotavirus vaccines also involve cells, virus seeds and biological reagents (such as growth supplements, serum, trypsin and any virus stabilizers used in the final product). Hence, each of these vaccine source components must be shown to be free from adventitious agents. The full passage history of the seed materials used for vaccine development should identify all substrates through which they have been passed to aid the development of appropriate programmes of testing for adventitious agents.viral seed lots should be assessed for absence of adventitious agents from all species that they may have been exposed to from isolation, through passage, and during production, including those that may be present in the raw materials used at each of these stages. The testing required will depend on available documentation on cell substrate donor, virus seed passage and derivation history and the seed virus may require purification (e.g. molecular cloning and plaque purification), if passage and derivation history is uncertain, or if it is contaminated with a known agent. 137

6 A risk assessment for transmissible spongiform encephalopathies would need to be included for the seed materials. The revised WHO Guidelines on transmissible spongiform encephalopathies in relation to biological and pharmaceutical products (5) provide guidance on risk assessments for master and working seeds and should be consulted. Many candidate vaccines are being developed in Vero cells. WHO has set a limit of 10 ng of DNA per human dose for parenterally administered vaccines based upon a WHO risk assessment (6) that established a safety factor. For orally delivered vaccines, the acceptable limit of residual cellular DNA has not been previously established. However, recent studies in rats, which compared the uptake efficiency of Vero cell DNA following administration by the oral and intramuscular routes indicated that Vero cell DNA given orally could be found in rat tissues at a concentration of fold less than when equivalent amounts of Vero DNA were given parenterally (7). Similarly, experiments in mice using polyoma DNA also showed a difference in uptake between the oral and parenteral routes (8). These preliminary data and the lack of evidence that cellular DNA is tumorigenic suggest that a DNA level of 100 µg/dose in an orally administered vaccine is equivalent to 10 ng in parenterally administered vaccines. In addition to the quantity of DNA, the size distribution of DNA should be evaluated during assessments of manufacturing consistency and process validation. It may be desirable for manufacturers to include steps in the process to reduce the level of high-molecular-weight cellular DNA. The national regulatory authority may require the further purification of virus harvests derived from continuous cell lines to remove cellular DNA, and/or the use of DNase treatment to reduce the size of DNA fragments. If the virus harvests are derived from diploid cell cultures, further purification is not required. The concentration of virus can be determined by infectivity titrations, e.g. plaque-forming or focus-forming assays. However, confirming the quantity of each rotavirus serotype in the final mixture of a multivalent, as opposed to a single component, vaccine is challenging. Conventional infectivity titrations require an antibody specific to each of the vaccine components. Molecular methods of measuring virus concentration that do not require the use of specific antibody, such as quantitative real-time polymerase chain reaction (PCR), have been developed and are being evaluated. One method uses Vero cell monolayers which are seeded in 96-well plates and inoculated with dilutions of a multivalent reference standard and samples. Viral replication is allowed to proceed for hours and the cells are then lysed by addition of Triton and one freeze thaw cycle. Cell lysates are assayed by real-time quantitative reverse transcriptase-pcr (QPCR) and rotavirus nucleic acid is quantified (9). Individual QPCR reactions are performed employing primer/probe sets specific to each of the reassortant 138

7 strains. Virus concentration is determined by parallel line analysis between the reference standard and each sample. International titration standards do not yet exist and WHO is considering developing such reagents and their subsequent characterization by international collaborative study. It should be borne in mind however that the differences in the properties of the candidate vaccines in terms of their attenuation may mean that such reference material will be product-specific. Manufacturers should set aside a preparation for use as an in-house standard. The real-time stability of each serotype in the final vaccine formulation should be measured to verify that the infectivity titre is at or above that level of virus required for vaccine efficacy at the end of the product shelflife. The thermal stability of each individual serotype should be determined in an appropriate stability study programme. It is advisable to assign a shelf-life to the intermediates that are intended to be stored on the basis of stability studies. WHO is developing further guidance on this issue. Based on the results of the stability testing programme, an accelerated stability test should be done on each new batch of vaccine. If the final product is mixed with vaccine diluent prior to use, the stability of this combination should also be determined. The consistency of a manufacturing process for live virus vaccine can best be determined by monitoring the content and quality of virus produced throughout the process, as well as the level of impurities following processing and the stability of the final product. Manufacturers should select relevant parameters based upon the specific production process used and develop methods to establish a programme to assure the consistency of production process. For example, the amount of virus produced by a standardized production process and the amount lost during processing should be consistent; the quality of the virus produced can be assessed by its thermal stability profile; the stability of the genomic sequence through multiple cell culture passages may be evaluated; process impurities and residuals such as residual host cell protein, residual cellular DNA, endotoxin, bovine serum, trypsin and antibiotics can be measured and their reduction during processing can be monitored. If the consistency of production has been validated, the monitoring programme could be conducted based on a reduced frequency of testing, but not on a batch-to-batch basis, with the agreement of the national regulatory authority. Special considerations Use of primary cells for the production of rotavirus vaccine The majority of live attenuated rotavirus vaccines that have been developed or are currently under development are produced in continuous cell lines 139

8 or diploid cells which are based on the cell bank system, so the main focus of these guidelines is on vaccines produced under such conditions. But the vaccine may also be produced in primary cells, in which case, the manufacturers should consult additional relevant guidelines or documents to ensure the safety of the final product. However, as suitable alternative cell substrates become available, primary cell cultures are less likely to be used in the future for many reasons (6).The following text provides brief guidance on issues that need to be considered if primary cells are used for rotavirus vaccine production, but there are some specific issues relating to the production of rotavirus vaccines in primary cell substrates which are not covered in these guidelines. If vaccine is produced in primary cells obtained directly from trypsinized tissue of normal healthy animals, such animals should be of a species approved by the national regulatory authority and not have been previously employed for experimental purposes. However, primary cell cultures prepared from wild animals show a high frequency of viral contamination. The number of viruses isolated and the frequency of isolation depend on many factors, including methods of isolation, test cell systems used, number of passages, duration of incubation and co-cultivation, and are directly proportional to the duration of the incubation period of the cultures. The frequency of cell culture contamination can be reduced by careful screening of the source animals to be used in production for the absence of antibodies to the relevant viruses. The use of animals bred in a carefully controlled colony, especially those which are specific-pathogen free, is strongly recommended. In addition, the use of secondary or tertiary cells on which testing for adventitious agents could be performed will reduce the frequency of contamination of the production cell culture. Production of uninfected control cultures and the extensive testing required for relevant adventitious agents becomes more challenging when producing live rotavirus vaccines in primary cells. A live attenuated rotavirus vaccine prepared from primary calf kidney cells has been approved for use in one country. There are no existing WHO guidelines for the production of vaccines on primary calf kidney cells. The WHO Requirements for the use of animal cells as in vitro substrates for the production of biologicals (6) provide general recommendations. The WHO Recommendations for the production and control of poliomyelitis vaccine (Oral) (10), Guidelines for the production and control of Japanese encephalitis vaccine (live) for human use (11) and Guidelines for the production of candidate live attenuated dengue virus vaccines (12) provide guidance on the use of primary cell substrates of monkey, hamster and dog kidney, respectively, and illustrate the principles that should be followed for vaccines produced in primary cell substrates from other species. 140

9 Part A. Guidelines on manufacturing A.1 Definitions A.1.1 International name and proper name The international name should be Live attenuated rotavirus vaccine (LARV) (oral) with additions to indicate the virus serotype(s) of the vaccine. The proper name should be equivalent to the international name in the language of the country of origin. The use of the international name should be limited to vaccines that satisfy the specifications formulated below. A.1.2 Descriptive definition Live attenuated rotavirus vaccine (oral) is a sterile vaccine preparation containing one or more virus serotypes, which have been grown through a seed lot system, prepared in a suitable approved cell substrate, formulated in a form suitable for oral administration and satisfying all the recommendations formulated in this document. At least five types of live attenuated rotavirus vaccine have been developed and/or are under development: a candidate rhesus rotavirus strain live attenuated vaccine; a candidate human-rhesus rotavirus strain live attenuated vaccine; a human rotavirus strain live attenuated vaccine; a candidate human bovine reassortant rotavirus strain live attenuated vaccine; a lamb rotavirus strain live attenuated vaccine. The preparation should satisfy all of the specifications formulated in these guidelines. Live attenuated rotavirus vaccine with an appropriate stabilizer may be freeze-dried or liquid. A.1.3 International reference preparations No international reference preparations are available at the time of preparing this document. A.1.4 Terminology The definitions given below apply to this document only. Candidate vaccine. A vaccine under development which is used in human clinical trials to assess its safety and efficacy. Virus master seed lot. A quantity of virus of uniform composition derived from an original isolate, processed at one time and passaged for a documented number of times. 141

10 Virus working seed lot. A quantity of virus of uniform composition derived from the master seed lot by a limited number of passages by a method approved by the national regulatory authority and fully characterized. The virus working seed lot is used for production of vaccine. Cell seed. A quantity of well-characterized cells, derived from a single tissue or cell of human, animal or other origin, stored frozen in liquid nitrogen in aliquots of uniform composition, one or more of which may be used for the production of a master cell bank. Cell bank. A collection of ampoules containing material of uniform composition stored under defined conditions, each ampoule containing an aliquot of a single pool of cells. Master cell bank (MCB). A quantity of fully characterized cells of human, animal or other origin stored frozen in liquid nitrogen in aliquots of uniform composition, derived from the cell seed. The master cell bank is itself an aliquot of a single pool of cells generally prepared from a selected cell clone under defined conditions, dispensed into multiple containers and stored under defined conditions. The master cell bank is used to derive all working cell banks. The testing performed on a replacement master cell bank (derived from the same cell clone, or from an existing master or working cell bank) is the same as for the initial master cell bank, unless a justified exception is made. Working cell bank (WCB). A quantity of cells of uniform composition derived from one or more ampoules of the master cell bank at a finite passage level, dispensed in aliquots into individual containers appropriately stored, usually stored frozen in liquid nitrogen, one or more of which would be used for production of cell culture. All containers are treated identically and once removed from storage, are not returned to the stock. Production cell culture. A cell culture derived from one or more ampoules of the WCB or primary tissue used for the production of vaccines. Adventitious agents. Contaminating microorganisms of the virus seed or cell substrate or materials used in its culture, which may include bacteria, fungi, mycoplasmas, and endogenous and exogenous viruses. Single harvest. A virus suspension of one virus type harvested from cell cultures prepared from a single production run. Monovalent virus pool. A homogenous pool of a number of single harvests of the same virus serotype, collected into a single vessel before clarification. Final bulk. The finished biological preparation present in the container from which the final containers are filled. The final bulk may be prepared from one or more clarified monovalent virus pools and may contain one or more virus serotypes. 142

11 Filling lot (fi nal lot). A collection of sealed final containers of vaccine that are homogeneous with respect to the risk of contamination during the filling process or freeze-drying. A filling lot must therefore have been filled or prepared in one working session. Focus forming unit (FFU). Viral infectivity identified on cell monolayers using rotavirus-specific antiserum. Plaque forming unit (PFU). The smallest quantity of a virus suspension that will produce a plaque in monolayer cell cultures. Cell culture infective dose 50% (CCID 50 ). The quantity of a virus suspension that will infect 50% of cell cultures. Unit of infectivity (UI). Relative viral infectivity of a sample inoculated in 96-well Vero cell monolayers measured by QPCR against a defined reference standard preparation. A.2 General manufacturing recommendations The general manufacturing requirements contained in Good manufacturing practices for biological products (13) should apply to establishments manufacturing oral rotavirus vaccine, with the addition of the following: Production steps and quality control operations involving manipulations of live rotavirus should be conducted under Biosafety Level 2 (14). Separate areas or a campaigned programme for the manufacturing of different virus serotypes are required. In production areas used for bulk formulation and filling, multiple serotypes may be present at the same time and these production areas may be campaigned with other vaccines provided sufficient cleaning validation and product changeover data is provided. A.3 Control of source materials A.3.1 Cell cultures for virus production A Master cell bank and working cell bank The use of a continuous cell line such as Vero cells (low passage, nontumorigenic phenotype) or diploid cells such as fetal rhesus lung cells, FrHL-2 (well-defined non-tumorigenic phenotype) for the manufacture of rotavirus vaccines should be based on the cell bank system. The cell substrates and cell banks should conform with the WHO Requirements for use of animal cells as in vitro substrates for the production of biologicals (6), as appropriate to continuous cells and diploid cells, and should be approved by the national regulatory authority. The maximum number of passages (or population doublings) allowable between the cell seed and the WCB and the 143

12 production cells should be approved by the national regulatory authority. Additionally, for Vero cells, the MCB or WCB cells should be propagated to or beyond the maximum production level and be examined for the presence of retroviruses and tumorigenicity in an animal test system (6). WHO has established a cell bank of Vero cells characterized in accordance with the requirements in the forty-seventh report of the WHO Expert Committee on Biological Standardization (6), which is available as a wellcharacterized source material (15) to manufacturers for preparation of their own MCB and WCB on application to the Coordinator, Quality Assurance and Safety of Biologicals, WHO, Geneva, Switzerland. The master cell bank, which is made in sufficient quantities and stored in a secure environment is used as the source material to make manufacturer s working cell banks. In normal practice a master cell bank is expanded by serial subculture up to a passage number (or population doubling, as appropriate) selected by the manufacturer and approved by the national regulatory authority, at which point the cells are combined to give a single pool distributed into ampoules and preserved cryogenically to form the WCB. The manufacturer s WCB is used for the preparation of production cell culture, and thus for production of vaccine batches. A Cell culture medium Serum used for the propagation of cells should be tested to demonstrate freedom from bacteria, fungi and mycoplasmas, according to the requirements given in Part A, sections 5.2 and 5.3 of the General requirements for the sterility of biological substances (16), as amended in 1995 and from infectious viruses. Suitable tests for detecting viruses in bovine serum are given in Appendix 1 of the WHO Recommendations for production and control of poliomyelitis vaccine (Oral) (10). Validated molecular tests for bovine viruses may replace the cell culture tests of bovine sera. As an additional indicator of quality, sera may be examined for freedom from phage and endotoxin. Irradiation may be used to inactivate potential contaminant viruses. The acceptability of the source(s) of any components of bovine, porcine, ovine or cervine origin used should be approved by the national regulatory authority. These components should comply with current WHO guidelines in relation to animal transmissible spongiform encephalopathies (5). If trypsin is used for preparing cell cultures and aiding in virus infection, it should be tested and found free of cultivable bacteria, fungi, mycoplasmas and infectious viruses, especially bovine or porcine parvoviruses, as appropriate. 144

13 The methods used to ensure this should be approved by the national regulatory authority. The trypsin should be gamma irradiated if possible. Human serum should not be used. If human albumin is used, it should meet the revised Requirements for the collection, processing and quality control of blood, blood components and plasma derivatives (Requirements for Biological Substances No. 27) (17), as well as current guidelines in relation to human transmissible encephalopathies (5). Penicillin and other beta-lactams should not be used at any stage of the manufacture. Other antibiotics may be used at any stage in the manufacture provided that the quantity present in the final product is acceptable to the national regulatory authority. Nontoxic ph indicators may be added, e.g. phenol red at a concentration of 0.002%. Only substances that have been approved by the national regulatory authority may be added. A Tests on master and working cell banks Tests on the MCB and WCB are performed in accordance with WHO Requirements for use of animal cells as in vitro substrates for the production of biologicals (6, 15). It is important to show that the cell banks are free of adventitious agents relevant to the species used in its derivation. Cell banks should be assessed for absence of adventitious agents that may have been present during production, including those that may be present in the source materials used at each of these stages. Full characterization may be performed on either the MCB or on the WCB (6, 15). A.3.2 Virus seeds A Virus strains Virus strains of rotavirus used for master and working seed lots to produce vaccines have in some cases been derived by genetic reassortment of animal rotavirus with human rotavirus with the desired serotypes or in other cases by multiple passages of human rotavirus in cell culture. The seed lot viruses should comply with the specifications of this section. Viruses may be passed in continuous, diploid, and/or primary cell lines. The candidate vaccine strains should be approved by the national regulatory authority. The strains of rotavirus used in the production of candidate rotavirus vaccines should be identified by historical records, which will include information on the origin of each strain, method of attenuation, whether the strains have been biologically cloned prior to generation of the master seed lots, genetic sequence information and the passage level at which attenuation for humans was demonstrated by clinical trials. 145

14 The vaccine production strain(s) should have been shown by appropriate laboratory tests (see A.3.2.3) and/or clinical studies to yield vaccines that are safe and efficacious in humans. Only strains approved by the national regulatory authority should be used. The immunogenicity of the virus strains, based upon the quantity of infectious virus of each serotype present in the vaccine that induces seroconversion when susceptible individuals are immunized with the vaccine, should be established in a dose response study. Any potential interference or potentiation between the serotypes in an infectivity assay should be evaluated prior to establishing this value. The immunizing dose established in this way serves as a basis for establishing parameters for potency at the time of release, stability and expiry date. A Virus seed lot system The production of vaccine should be based on the virus master seed lot and virus working seed lot system. Seed lots should be prepared in the same type of cells and by the same production process as those used for production of final vaccine. Virus seed lots should be stored in a dedicated temperature-monitored refrigerator at a temperature that ensures stability and the storage arrangement should ensure appropriate security of the virus seed lots. Seed lots of rotavirus used in the production of live attenuated rotavirus vaccine (oral) should be identified by historical records, which should include information on their origin. Only virus seed lots that are approved by the national regulatory authority should be used (see General considerations). The virus master seed lot is generally produced from a biological clone of the attenuated parent virus (animal/human reassortant or cell-culture passaged human virus) and is made in sufficient quantities and stored in a secure environment and is used as the source material to make the manufacturer s virus working seed lots. Either the virus master seed lots or the virus working seed lots should be fully characterized and be tested extensively for adventitious agents, and approved by the national regulatory authority. The virus master seed lot also serves as a benchmark against which to compare virus produced by subsequent passage in cell culture or shed virus following vaccination. The manufacturer s virus working seed lot is used for the production of vaccine batches and is prepared from the master virus seed lot. It is recommended that a large lot of virus working seed be set aside as the basic material that the manufacturer should use for the preparation of each batch of vaccine. The virus working seed lot should be prepared by defined number of passages from the virus master seed lot by a method and a passage level from the original virus seed that is established through clinical and 146

15 vaccine development studies. Once the passage level of the working seed lot is established, it may not be changed without approval from the national regulatory authority. A Tests on virus master and working seed lots A Identity Each seed lot should be identified by virus type by an immunological assay or by sequencing. A molecular identity test, electropherotyping, is one recognized method for identifying rotaviruses. Comparing human and animal rotaviruses and differentiating between subgroups can be achieved through electropherotyping. Electropherotyping can be used as a means for identifying gross alterations in genomic differences between the parent wild type viruses and/or animal donor virus (if employed) and the vaccine strains. The identity of the vaccine virus is confirmed by comparing the electrophoretic profile of the vaccine virus to the RNA electrophoretic profile of a known human rotavirus serotype. Other molecular identity tests may include RNA/RNA hybridization and enzyme restriction maps or genetic sequences of PCRamplified VP7 gene segments. The tests should be approved by the national regulatory authority. A Genotype/phenotype characterization Genotype and phenotype stability of the seed lots upon passage should be measured using relevant assays to ensure uniformity of vaccine lots. Genetic characterization of the viruses has played a major role in the development of the vaccines already licensed or currently nearing licensure. It helps to assess the genetic and phenotypic stability of the seed lots on passage, and molecular methods have been used intensively. Other approaches including in vitro phenotypic markers might be considered. Genetic characterization of the seed lot viruses has included determination of the nucleotide sequence of the complete viral genome, analysis of the molecular basis of the attenuated phenotype and determination of the genetic stability of the virus seed lots by comparison of the nucleotide sequence of the viral genome at different passage levels. It should be noted that full-length sequencing may not identify minority populations of variants that may be present in vaccines. In some studies the entire genomic sequence of all 11 gene segments of the virus master seed lot has been sequenced and compared with the sequence of the virus working seed lot, vaccine production lots and vaccine virus shed in stools of vaccinees. If the attenuation loci are known, sequencing around these sites can be performed instead of on the entire genome. Given the frequency of errors of the RNA replicating polymerase, base changes are likely to be found. The potential impact of any changes observed (silent or in protein-coding regions) should be evaluated. 147

16 Specific assays for markers of attenuation are still in development and the manufacturer should make an effort to investigate appropriate assays that are relevant to their vaccine. A Tests for bacteria, fungi and mycoplasmas Each virus seed lot should be tested for bacterial, fungal and mycoplasmal contamination by appropriate tests as specified in Part A, sections 5.2 and 5.3, of the General requirements for the sterility of biological substances, as amended in 1995 (16). A Tests for adventitious viruses Each virus seed lot should be tested in cell cultures for adventitious viruses appropriate to the origin and the passage history of the seed virus (18). Neutralization of rotavirus is necessary for many tests because the virus is cytopathogenic. Antisera used for this purpose should be shown to be free from antibodies that may neutralize specific adventitious viruses being tested for. If neutralization of rotavirus is not possible, the test sample may be passaged in trypsin-free media prior to initiating the assay, to reduce the ability of rotavirus to infect the indicator cell substrates. The cells inoculated should be observed microscopically for cytopathic changes. At the end of the observation period, the cells should be tested for haemadsorbing viruses. Each master or working seed lot should also be tested in animals that may include guinea-pigs, mice and embryonated chicken eggs, as appropriate. For test details refer to the WHO Requirements for measles vaccines (Live) (19), and the European Pharmacopoeia, 2002 (18) Additional testing for specific adventitious viruses may be performed, for example, using PCR amplification techniques. A Virus concentration Each seed lot should be assayed for infectivity in a sensitive assay in a cell culture system. An immunofocus or plaque forming assay may be used in MA-104, Vero or other sensitive cells to determine virus concentration. The assay is based on the visualization of infected areas (plaques or focus of infection) of a cell monolayer directly or by probing with rotavirus-specific antibodies. Results should be recorded as focus-forming units (FFU/ml) or plaque-forming units (PFU/ml). A cell culture infectious dose assay may also be used to determine virus concentration. Results should be recorded as CCID 50 /ml. Alternatively, quantitative PCR detection of virus replication in a cell culture system may be used to provide an appropriate measure of infectivity. Results should be recorded as units of infectivity (UI/ml). 148

17 The detailed procedures for carrying out the tests and for interpreting the results should be those approved by the national regulatory authority. A.4 Control of vaccine production A.4.1 Control of cell cultures From the cell suspension used to prepare cell cultures for growing attenuated rotavirus, an amount of processed cell suspension equivalent to at least 5% or 500 ml of cell suspension, whichever is greater, should be used to prepare control cultures of uninfected cells. If fermenter technology is used, the size and treatment of the cell sample to be examined should be approved by the national regulatory authority. These control cultures should be observed microscopically for changes attributable to the presence of adventitious agents for at least 14 days after the day of inoculation of the production cultures or at the time of final virus harvest if this is later. The control cultures should be incubated under essentially similar conditions to those used for the production cultures with agreement of the national regulatory authority. At the end of the observation period, fluids collected from the control culture from each single virus harvest should be tested for the presence of adventitious agents as described below (A.4.1.2). Samples that are not tested immediately should be stored at 60 ºC or below. If any test shows evidence of the presence of adventitious agents in control cultures, the harvest of virus from these cultures should not be used for vaccine production. For the test to be valid, not more than 20% of the control culture vessels should have been discarded for nonspecific accidental reasons by the end of the test period. A Tests for haemadsorbing viruses At the end of the observation period, cells comprising no less than 25% of the control cells should be tested for the presence of haemadsorbing viruses, using guinea-pig red blood cells. If the red blood cells have been stored, the duration of storage should not have exceeded 7 days, and the temperature of storage should have been in the range of 2 8 ºC. In some countries, the national regulatory authority requires that additional tests for haemadsorbing viruses be performed using other species of red blood cells including those from humans (blood group O), monkeys and chickens (or other avian species). In all tests readings should be taken after incubation for 30 minutes at 0 4 ºC, and again after a further incubation for 30 minutes at ºC. A further reading for the test with monkey red blood cells should be taken after an additional incubation for 30 minutes at ºC. 149

18 For the tests to be valid, not more than 20% of the culture vessels should have been discarded for nonspecific accidental reasons by the end of the test period. A Tests for other adventitious agents At the end of the observation period, a sample of the pooled fluid from each group of control cultures should be tested for adventitious agents. For this purpose, 10 ml of each pool should be tested in the same cells, but not the same batch of cells, as those used for the production of virus, and additional 10-ml samples of each pool should be tested in human cells and at least one other sensitive cell system. Each sample should be inoculated into bottles of these cell cultures in such a way that the dilution of the pooled fluid in the nutrient medium does not exceed 1 in 4. The area of the cells should be at least 3 cm 2 per ml of pooled fluid. At least one bottle of each kind of cell cultures should remain uninoculated to serve as a control. The inoculated cultures and control cultures should be incubated at a temperature of ºC and should be observed for cytopathic effects for a period of at least 14 days. For the tests to be valid, not more than 20% of the culture vessels should have been discarded for nonspecific accidental reasons by the end of the test period. Some national regulatory authorities require that, at the end of this observation period, a subculture is made by inoculating the fluid onto fresh cells in the same culture system and observed for at least 7 days. Furthermore, some national regulatory authorities require that these cells should be tested for the presence of haemadsorbing viruses. A Identity test At the production level, the cells should be identified by means of tests approved by the national regulatory authority. Suitable methods are, but are not limited to, biochemical tests (e.g. isoenzyme analyses), immunological tests (e.g. HLA assays), cytogenetic tests (e.g. for chromosomal markers), and tests for genetic markers (e.g. DNA fingerprinting). A.4.2 Cell cultures for vaccine production A Tests for adventitious agents On the day of inoculation with the virus working seed lot, each cell culture and/or cell culture control should be examined for degeneration caused by infectious agents. If such examination shows evidence of the presence in a cell culture of any adventitious agents, the culture should not be used for vaccine production. Prior to infection, samples of each cell culture are removed for sterility and mycoplasma testing. 150

19 If animal serum is used for cell cultures before the inoculation of virus, it should be removed and replaced with serum-free maintenance medium, after the cells have been washed with serum-free medium, if appropriate. A Tests for bacteria, fungi and mycoplasmas A volume of at least 20 ml of the pooled supernatant fluids from the production cell culture should be tested for bacterial, fungal and mycoplasmal sterility by appropriate tests as specified in Part A, sections 5.2 and 5.3, of the General requirements for the sterility of biological substances, as amended in 1995 (16). A.4.3 Control of single harvests and monovalent virus pools A Virus inoculation Cell cultures are inoculated with rotavirus working seed at a defined multiplicity of infection. After viral adsorption, cell cultures are fed with maintenance medium and incubated within a defined temperature range and for a defined period of time, usually established based upon the degree of cytopathic effect. The range of multiplicity of infection, temperature, ph and time period of incubation will depend on the vaccine strain and production, a defined range should be established by the manufacturer and be approved in the marketing authorization by the national regulatory authority. A Monovalent virus pools A virus single harvest is harvested within a defined time period postinoculation. A monovalent virus pool may be the result of one or more single harvests (from multiple tissue culture flasks, cell factories or bioreactors) in which all harvests were derived from one or a small number of ampoules of the WCB and the same virus working seed lot recovered at the same time. If multiple single harvests are taken, each single harvest should be sampled for testing, stabilized and stored under suitable conditions until pooling. No antibiotics should be added at the time of harvesting or at any later stage of manufacture. Samples of monovalent virus pools should be taken for testing and stored at a temperature of 60 ºC or below. A Tests on single harvest or monovalent virus pools Tests may be done on single harvest or on virus pool. If the tests are done on the virus pool, the protocol should be approved by the national regulatory authority. A Sampling Samples required for the testing of virus harvests should be taken immediately on harvesting prior to further processing. If the tests for adventitious agents 151

20 as described in Part A, section A , are not performed immediately, the samples taken for these tests should be kept at a temperature of 60 ºC or below and subjected to no more than one freeze thaw cycle. A Identity Each single harvest or virus pool should be identified as the appropriate rotavirus serotype by immunological assay or by a molecular-based assay, e.g. electropherotyping, RNA/RNA hybridization or PCR. The tests should be approved by the national regulatory authority. A Tests for bacteria, fungi and mycoplasmas Each single harvest or virus pool should be shown to be free from bacterial, fungal and mycoplasmal contamination by appropriate tests as specified in Part A, sections 5.2 and 5.3, of the General requirements for the sterility of biological substances, as amended in 1995 (16). A Tests for adventitious agents For the purposes of the recommendations set out in this section of Part A, the volume of each single harvest or virus pool taken for neutralization and testing should be at least 10 ml and should be such that a total of at least 50 ml or the equivalent of 500 doses of final vaccine, whichever is the greater, has been withheld from the corresponding final bulk. Each virus pool should be tested in cell cultures for adventitious viruses appropriate to the passage history of the seed virus (18). Neutralization of rotavirus is necessary for many tests because the virus is cytopathogenic. Antisera used for this purpose should be shown to be free from antibodies that may neutralize the adventitious viruses being tested for. If neutralization of rotavirus is not possible, the test sample may be passaged in trypsinfree media prior to initiating the assay to reduce the ability of rotavirus to infect the indicator cell substrates. The cells inoculated should be observed microscopically for cytopathic changes. At the end of the observation period, the cells should be tested for haemadsorbing viruses. Additional testing for specific adventitious viruses may be performed, for example using PCR amplification techniques. A Virus concentration Each virus pool should be assayed for infectivity using a sensitive assay in cell cultures to monitor the consistency of production. An immunofocus or plaque assay may be used in MA-104, Vero or other sensitive cells to determine virus concentration. The assay is based on the visualization of infected areas of a cell monolayer directly or by probing 152